Pathogenesis can be viewed as a strategic response of bacteria to environmental conditions they encounter in a human or animal host. For many pathogens, a key environmental signal is nutrient availability. CodY, a protein found in nearly all low-G+C Gram-positive bacteria, is a key factor that regulates major metabolic pathways and virulence gene expression in response to nutrient availability in Staphylococcus, Streptococcus, Clostridium, Bacillus and Listeria. CodY is activated by GTP (except in Streptococcus) and the branched-chain amino acids (isoleucine, leucine and valine; BCAAs). When the intracellular pools of these effectors decrease, CodY generally loses activity and genes that it represses are turned on. Our recently acquired evidence indicates that L. monocytogenes CodY is necessary for expression and activity of the major virulence regulator, PrfA. Surprisingly, CodY activates prfA transcription by binding within the prfA coding sequence when BCAAs are limiting. This proposal aims to unravel the mechanistic basis for the counterintuitive role of CodY in prfA regulation, while at the same time placing such regulation in the context of the global role of CodY. That is, the hundreds of genes that respond to changes in CodY activity are expected to be expressed in a hierarchical manner as the intracellular pools of the CodY effectors change. The analysis of the hierarchy will provide insight into the bacterium's strategy for using particulr metabolic pathways ahead of others and for expressing different virulence genes at different levels of nutrient availability. In addition, the BCAAs have been shown to have a complex role in pathogenesis. They are not only key components of proteins and effectors of CodY activity, but are also the precursors for the branched-chain fatty acids (BCFAs) that are the major constituents of L. monocytogenes membranes. Substituting straight-chain fatty acids for the BCFAs makes L. monocytogenes much less virulent by a mechanism that involves reducing PrfA activity. Since CodY represses BCAA biosynthesis and activates prfA transcription, understanding the mechanism by which CodY brings about the appropriate level of PrfA activity will give important new insight into the integration of metabolism and virulence as well as the infection process as a whole.